Heterotopic ossification (HO) is a frequent complication in patients who have suffered head and neck traumas, traumatic acetabular fracture, or undergone total hip replacement. It is a process of bone formation at ectopic sites, such as muscle and connective tissue, that can lead to decreased mobility, pain, or even total ankylosis, predominantly in hip and elbow joints (for review see: Eulert, J., et al., Der Unfallchirurg 100 (1991) 667-674; Nilsson, O. S., Acta Orthop. Scand. 69 (1998) 667-674). As the number of elderly people increases and, subsequently, the number of the total hip arthoplastic operations rises, the number of patients suffering from HO can be expected to grow. HO is also manifested in some inherited diseases, such as fibrodysplasia, or acquired bone forming lesions, such as spinal hyperostosis, myelopathy and spondylitis ankylosans, in which no curable treatment is available or a surgical operation is the only means of treatment.
Effective prevention of the formation of extra bone following total hip replacement is needed in about 10 to 50% of patients (Seegenschmiedt, M. H., et al., Int'l. J. Radiation Oncology Biol. Phys. 39 (1997) 161-171) and presently radiotherapy, non-steroidal anti-inflammatory drugs or both are being used for prevention (Coventry M. B. and Scanion P. W., J. Bone Joint Surg. 63 (1981) 201-208; Dahl, H. K., in Symposium on Arthrose, Ed. MSD Blinderm, Norway 1975, pp. 37-46). Unfortunately, these treatments are associated with potential side effects, which restrict their application to high-risk patients only (Nielsson, O. S., supra).
A group of proteins, designated as bone morphogenetic proteins (BMPs), which occur naturally as dimers, play a key role in any ossification process. The BMP-family is divided to subfamilies including the BMPs, such as BMP-2 and BMP-4, osteogenic proteins (OPs), such as OP-1 or BMP-7, OP-2 or BMP-8, BMP-5, BMP-6 or Vgr-1, cartilage-derived morphogenetic proteins (CDMPs), such as CDMP-1 or BMP-14 or GDF-5, growth/differentiation factors (GDFs), such as GDF-1, GDF-3, GDF-8, GDF-9, GDF-11 or BMP-11, GDF-12 and GDF-14, and other subfamilies, such as BMP-3 or osteogenin, BMP-9 or GDF-2, and BMP10. The BMP-family with more than thirty members belongs to the TGF-β-super-family (Reddi, H., Cytokine & Growth Factor Reviews 8 (1997) 11-20).
The overall folding topology of the BMP-family members and other members of the TGF-β super-family, for which the 3-dimentional structure has been determined, resembles a hand exhibiting the victory sign, with the wrist representing the central alpha-helix, two fingers, two anti-parallel β-sheets, and the palm the cysteine-knot region (Scheufeler, C. et al., J. Mol. Biol. 287 (1999) 103-115). The cysteine-knot consists of three intrachain disulfide bridges and is the main stabilizer of the 3-D structure, and it is so effective that the biological activity is preserved even after extensive exposure to low pH, urea or guanidine hydrochloride treatments (Sampath, T. K., and Reddi, A. H., Proc. Natl. Acad. Sci. USA 78 (1981) 7599-7603). Further stabilization of the topology is achieved by the dimerization of two monomers, creating an internal hydrophobic core (Scheufeler, C. et al., supra). The overall dimeric structure is also necessary for the biological action, such as osteoinduction, as it enables the simultaneous binding of a dimeric molecule to the corresponding receptor, for instance the simultaneous binding of a BMP-dimer to a type I and a type II serine/threonine receptor, forming a heterotetramer which triggers a signal cascade via the phosphorylation of Smads (Kawabata, M. et al., Cyto & Growth Factor Rev. 9 (1998) 49-61; Massaqué, Annu. Rev. Biochem. 67 (1998) 753-791). Recently, it has been shown that BMP-2 dimers can exist as two biological distinct folding variants, an osteoinductive (os)BMP-2 and an osteoinhibitive (inh)BMP-2, which differ only in their disulfide bridge composition (Weber, F., et al., Biochem. Biophys. Res. Commun. 286 (2001) 554-558).
Results based on the protein and nucleotide sequences of the BMPs have revealed that the morphogenesis of bone, comprising chemotaxis, mitosis, and differentiation, is governed by the action of the BMPs. It has also been shown that the effects of the BMPs are not limited to bone and cartilage. In the early stage of embryogenesis the BMPs rule the formation of the entire body plan and specify the tissue types and axes. In an adult, the BMPs affect to the ability of bones to repair successfully (for review, see Wozney, J. and Rosen, V., Clin. Orthop. Rel. Res. 346 (1998) 26-37). This aspect of the BMP action together with its osteoinductive power suggests the use the BMPs in the treatment of patients for the enhancement of fracture healing and the augmentation of bone. Another aspect of the BMPs is their interaction in the manifestation of heterotopic ossification, which has created expectations to develop inhibitors of the BMPs and use them as therapeutic agents in HO.
BMP mutants lacking the ability to form the correct disulfide bond have been disclosed in Finnish Patent Application 20011478. Osteoinhibitory recombinant BMP-4 mutants spanning amino acids 48 to 116 of the 116 amino acids of the mature BMP-4 (Sequence Id. No. 2), i.e. covering the entire region involved in the formation of finger 2 plus the wrist epitope but not that of finger 1 (mutant 1), and BMP-4 mutants spanning amino acids 1 to 55 (mutant 2) or 1 to 88 (mutant 3), i.e. containing only the region for finger I, but not finger 2, are exemplified in said application. The cysteine responsible for dimerization is located at position 80 and is therefore present in mutants 1 and 3. The cysteine residues involved in the formation of the cysteine-knot are at positions 16, 45, 49, 81, 113 and 115 and accordingly, none of the mutants is capable of forming the entire cysteine-knot structure. It was found that mutant 1, but not mutants 2 or 3, was able to reduce alkaline phosphatase activity in MC3T3 E1 cells, while the presence or absence of cysteine 80, which is involved in dimer formation, did not correlate with the cellular response. It was therefore concluded that the amino acid sequence responsible for the inhibition of ossification resides in the C-terminal half of mature BMP, the region where an anti-parallel B-sheet, adopting a twisted crossover conformation plus the wrist, epitope form finger 2.
Additional and more effective inhibitors of the BMPs useful in the treatment of heterotopic ossification are still needed.
A purpose of the present invention is to provide additional novel means for the utilization of the bone forming inhibitory activity of the BMPs and other members of the TGF-β super-family, where applicable, in the treatment of patients in orthopaedics and other fields in medicine.
Specifically, a purpose of the present invention is to provide novel BMP mutants that would be useful in the treatment and prevention of heterotopic ossification and other diseases involving undesired bone formation. Such mutants would significantly add to the options that now are available in the treatment of HO, and would lack the side effects of the non-steroidal anti-inflammatory drugs (NSAIDs) and the radiotherapy, which at present are the alternative methods of treatment.